A Kinetic Characterization of (Na⁺, K⁺)-ATPase Activity in the Gills of the Pelagic Seabob Shrimp Xiphopenaeus kroyeri (Decapoda, Penaeidae)

Abstract

We characterize the kinetic properties of a gill (Na⁺, K⁺)-ATPase from the pelagic marine seabob Xiphopenaeus kroyeri. Sucrose density gradient centrifugation revealed membrane fractions distributed mainly into a heavy fraction showing considerable (Na⁺, K⁺)-ATPase activity, but also containing mitochondrial F₀F₁- and Na⁺- and V-ATPases. Western blot analysis identified a single immunoreactive band against the (Na⁺, K⁺)-ATPase α-subunit with an M_r of ≈110 kDa. The α-subunit was immunolocalized to the intralamellar septum of the gill lamellae. The (Na⁺, K⁺)-ATPase hydrolyzed ATP obeying Michaelis–Menten kinetics with V_M = 109.5 ± 3.2 nmol Pi min⁻¹ mg⁻¹ and K_M = 0.03 ± 0.003 mmol L⁻¹. Mg²⁺ (V_M = 109.8 ± 2.1 nmol Pi min⁻¹ mg⁻¹, K_0.5 = 0.60 ± 0.03 mmol L⁻¹), Na⁺ (V_M = 117.6 ± 3.5 nmol Pi min⁻¹ mg⁻¹, K_0.5 = 5.36 ± 0.14 mmol L⁻¹), K⁺ (V_M = 112.9 ± 1.4 nmol Pi min⁻¹ mg⁻¹, K_0.5 = 1.32 ± 0.08 mmol L⁻¹), and NH₄ ⁺ (V_M = 200.8 ± 7.1 nmol Pi min⁻¹ mg⁻¹, K_0.5 = 2.70 ± 0.04 mmol L⁻¹) stimulated (Na⁺, K⁺)-ATPase activity following site–site interactions. K⁺ plus NH₄ ⁺ does not synergistically stimulate (Na⁺, K⁺)-ATPase activity, although each ion modulates affinity of the other. The enzyme exhibits a single site for K⁺ binding that can be occupied by NH₄ ⁺, stimulating the enzyme. Ouabain (K_I = 84.0 ± 2.1 µmol L⁻¹) and orthovanadate (K_I = 0.157 ± 0.001 µmol L⁻¹) inhibited total ATPase activity by ≈50 and ≈44 %, respectively. Ouabain inhibition increases ≈80 % in the presence of NH₄ ⁺ with a threefold lower K_I, suggesting that NH₄ ⁺ is likely transported as a K⁺ congener.